The invention relates to special biologically active molecules, particularly based on peptide nucleic acids (PNA) and short interfering RNA (siRNA), a method for their transfection into a target cell and cell-specific activation in this cell or directly before their transfection, and an application kit to be administered in combination with a transfection system. Said biologically active molecules interact with the mRNA of the target gene and in the case of siRNA they form together with specific endoribonucleases an RNA protein complex named RISC (RNA induced silencing complex). The RISC complex bonds to the target mRNA and endonucleases restrict the target mRNA. In this way, gene expression is suppressed and consequently the formation of target proteins is inhibited. If activated PNA molecules are used, the translation will be prevented due to the bonding to the target mRNA.
The cell-specifically activatable, biologically active molecules can be used, for example, for combating abnormal cells and inhibiting their growth, particularly in tumor treatment, treatment of virus infections, and age-specific treatments for example. Generally, the cell-specifically activatable, biologically active molecules can be used for the modulation of gene expression of the target cells. This modulation does not only allow reduction of the gene expression but also increase thereof by achieving a reduction of the expression of the negative regulators of the target gene by means of the biologically active molecules.
The inhibition of gene expression by introducing short (19-23 bp), double-stranded RNA molecules (siRNA) or PNA molecules in eukaryotic cells, which is specific for a sequence segment of the mRNA of a target gene, has already been described (Elbashir S M et al.: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature, 2001 May 24, 411(6836), 494-8; Liu Y et al.: Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids, Biochemistry, 2004 Feb. 24, 43(7), 1921-7; U.S. Pat. No. 5,898,031; U.S. Pat. No. 7,056,704).
The use of such molecules does not prevent the reading of a gene and the generation of an mRNA, but in the case of siRNA it initiates an endogenous mechanism that degrades the target mRNA. Finally, as written above, the formation of a specific protein is suppressed without impairing the expression of further genes (post-transcriptional gene silencing).
To suppress the expression of a gene, the siRNA and PNA molecules can be directly introduced into the cell, particularly via transfection reagents and electroporation (Zhang M et al.: Downregulation enhanced green fluorescence protein gene expression by RNA interference in mammalian cells, RNA Biol. 2004 May, 1(1), 74-7; Gilmore I R et al.: Delivery strategies for siRNA-mediated gene silencing, Epub 2004 May 22., Curr Drug Deliv. 2006 Apr. 3(2), 147-5; U.S. Pat. No. 6,506,559).
The disadvantage of this method is the relative instability of the siRNA but it can be reduced by chemical modifications (U.S. Pat. No. 6,107,094).
A particular problem for the therapeutic application of biologically active molecules is an application in vivo. Methods for stabilizing the siRNA have been developed for such an application in order to reduce the degradation (Morrissey et. al.: Chemical Modifications of Synthetic siRNA, Pharmaceutical Discovery, May 1, 2005), and transfection reagents have been engineered, for example nanoparticles, in vivo-jetPEI™ (Polyplus), that introduce the siRNA into the cells in vivo, too (Vernejoul et al.: Antitumor effect of in vivo somatostatin receptor subtype 2 gene transfer in primary and metastatic pancreatic cancer models, Cancer Research 62, 2002, 6124-31; Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A: RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo, Gene Ther 12(5), 2005, 461-6.).
Furthermore, methods have been evolved to increasingly transfect siRNA into cells of a target tissue in vivo (Ikeda et. al.: Ligand-Targeted Delivery of Therapeutic siRNA, Pharmaceutical Research, Vol. 23, No. 8, August 2006).
However, the administration of biologically active substances in vivo is often problematic due to the systemic effect. The selective introduction of these substances into target cells is not sufficiently specific. This disadvantage is particularly important for siRNA and PNA molecules that shall selectively act and shall have this selective effect only in the target cells. The cell specificity achieved by transfection reagents that are provided with a tissue or cell marker (e.g. antibody/antigen-marked nanoparticles, TAT protein flanking and others) is not sufficiently high. Wrong transfection is the result.
Moreover, a method is known that deactivates the biological effect of siRNA molecules by bonding fluorochromes and bringing back said molecules to their active state by exposing them to light of a defined wave length (Q N Nguyen et al.: Light controllable siRNAs regulate gene suppression and phenotypes in cells, Biochim Biophys Acta, 2006). This activation is initiated from the outside and is not cell-specific in any way. After their activation said siRNA molecules have consequently an undesired effect in all the other transfected cells, too and not only in the target cells as intended.
Furthermore, it is also difficult to use this mechanism for in vivo applications.